Summary. Existing antiretroviral drugs do not clear HIV-1 latent reservoirs, underscoring the urgent need for new therapeutic strategies. The HIV-1 Nef accessory factor is an attractive target for drug development because of its critical roles in the HIV-1 life cycle and immune system escape. Our group has discovered novel small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in donor PBMCs. Most importantly, our Nef inhibitors rescue cell-surface MHC-I expression in latently infected, patient-derived CD4 T-cells, enabling recognition and killing by autologous CTLs. Thus, Nef inhibitors represent an innovative approach to antiretroviral therapy that may provide a path to eradication of HIV-1 reservoirs. Our renewal application is focused on the mechanism of action of our most promising class of inhibitors (hydroxypyrazoles) against Nef-mediated enhancement of HIV-1 infectivity, replication, and immune escape. Results of these studies will strengthen our drug development efforts targeting Nef as part of a functional cure strategy. Four Specific Aims are proposed: Aim 1. Identify Nef residues essential for inhibitor action by combining biophysical and genetic approaches. Preliminary and published data strongly suggest that hydroxy- pyrazole Nef inhibitors, which disrupt multiple Nef functions, may have a common mechanism of action involving suppression of Nef homodimerization or stabilization of non-functional Nef-effector complexes. Using a combi- nation of X-ray crystallography as well as Nef codon mutagenesis plus inhibitor selection, we will identify Nef amino acids essential for inhibitor action. Aim 2. Explore the mechanisms by which Nef inhibitors suppress HIV- 1 infectivity. Hydroxypyrazole Nef inhibitors reduce HIV-1 infectivity in TZM-bl reporter cells to the same extent as Nef-deleted viruses. This Aim will explore the whether Nef inhibitors restore virion incorporation of SERINC5, a well-known HIV-1 restriction factor linked to Nef. We will also pursue Nef inhibitor effects on overall virion protein composition using an unbiased proteomics approach, which has the potential to identify host cell factors that are uniquely incorporated (or excluded) by Nef inhibition. Aim 3. Test the role of Tec-family kinases in the suppression of HIV-1 replication by Nef inhibitors. HIV-1 Nef interacts with Itk and Btk, two members of the Tec kinase family expressed in HIV-1 host cells. Inhibition of Itk suppresses the HIV-1 life cycle, suggesting that Nef- mediated Itk activation supports efficient viral replication in CD4 T cells. We will explore the mechanism of Nef- induced Tec-family kinase activation, and whether Nef inhibitors potently block HIV-1 replication in T cells via the Nef-Itk pathway. Aim 4. Investigate the mechanism of Nef inhibitor action on MHC-I downregulation. This Aim will explore the effect of Nef inhibitors on crystal structures of Nef in complexes with the MHC-I cytoplasmic tail and the AP-1 1 subunit, interactions essential for immune escape. Inhibitor effects on Nef interactions with MHC-I/AP-1 will also be explored in cells using a bimolecular fluorescence complementation (BiFC) assay. These studies will clarify the mechanisms by which Nef inhibitors restore CTL responses to HIV infection.